Glycolipid compositions and methods of use

ABSTRACT

The invention provides immunostimulatory glycolipids and compositions thereof and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/362,630, entitled “GLYCOLIPIDCOMPOSITIONS AND METHODS OF USE,” filed Jul. 15, 2016, the entirecontents of which are incorporated by reference herein.

FIELD

The invention relates to glycolipids, compositions, methods ofsynthesis, isolation and/or purification, and methods of use thereof.

BACKGROUND

Polysaccharide A (PSA) of Bacteroides fragilis (B. fragilis) has beenreported to be an immunomodulator with therapeutic and prophylacticactivities. U.S. Pat. Nos. 5,679,654 and 5,700,787; Tzianabos A O et al.(2000) J Biol Chem 275:6733-40. As an example, PSA has been shown toprovide protection from intestinal inflammatory diseases as well assystemic immune-mediated diseases such as the mouse model for multiplesclerosis, experimental autoimmune encephalomyelitis (EAE). PSA is oneof eight capsular polysaccharides made by B. fragilis.

PSA was recently discovered to possess a lipid moiety. The lipid moietywas hypothesized to anchor the polysaccharide in the B. fragilis outermembrane. It was also recently discovered that this “lipidated PSA” wasmore potent than non-lipidated PSA (referred to herein as “PSA”) formsprovided in the prior art.

SUMMARY

The invention is based, in part, on the identification andcharacterization of the glycolipid moiety that is found conjugated toPSA using certain isolation methods. The invention is further premised,in part, on surprising finding that such glycolipid is able to stimulatethe innate immune response and in particular activate antigen-presentingcells such as plasmacytoid dendritic cells. Based on this surprisingfinding, this disclosure contemplates and provides, among other things,use of the glycolipids as adjuvants in for example vaccine formulations.

The innate immunostimulatory property of the glycolipid is enhanced uponcleavage of the glycolipid moiety from the polysaccharide chain of PSA.

The disclosure therefore provides, in some aspects, glycolipids ofdefined chemical structure, as well as compositions comprising suchglycolipids. Such compositions may be further defined by the purityand/or concentration of glycolipids contained therein.

Also provided are compositions of the glycolipids with antigens such asbut not limited to bacterial antigens, including without limitation PSAand lipidated PSA. In some instances, such compositions compriselipidated PSA and glycolipid in ratios ranging from 4:1 to 20:1 (w/w),or 4:1 to 15:1 (w/w), or 4:1 to 10:1 (w/w).

In one aspect, provided herein is a glycolipid comprising atri-acylated, tetra-acylated or penta-acylated diglucosamine, and thediglucosamine may be optionally conjugated to an oligosaccharide. Theglycolipid may be isolated intending that it is physically separatedfrom other components including contaminants such as but not limited tocomponents of B. fragilis cells. Isolation may also intend a degree ofpurity or concentration in a composition. The glycolipid unlessotherwise stated is unconjugated to the polysaccharide chain of PSA(i.e., the polysaccharide comprised of repeating tetrasaccharides foundin PSA and lipidated PSA). Thus, it is to be understood that if theacylated diglucosamine is conjugated to the oligosaccharide, sucholigosaccharide is not the PSA polysaccharide, and such glycolipid isnot PSA. The glycolipid may be formulated with such polysaccharide chainof PSA or it may be formulated with lipidated PSA or it may beformulated with another antigen or another polysaccharide bearingmoiety, although it will typically be provided in an unconjugated orfree form. Examples of such formulations include for example micelles orliposomes or other delivery vehicles that are amenable to lipid basedagents.

In some embodiments, the diglucosamine is tetra-acylated orpenta-acylated. In some embodiments, the tri-acylated, tetra-acylated orpenta-acylated diglucosamine comprises acyl chains ranging in lengthfrom 14-17 carbons. In some embodiments, the tri-acylated,tetra-acylated or penta-acylated diglucosamine comprises acyl chainsrange in length from 15-17 carbons.

In some embodiments, the glycolipid is substantially free of othercomponents found in a B. fragilis capsule or found in B. fragilis cells.In some embodiments, the glycolipid is substantially free of LPS. Insome embodiments, the glycolipid is substantially free of polysaccharideincluding for example the repeating tetrasaccharide polysaccharide ofPSA.

In some embodiments, the glycolipid is provided in purified form. Insome embodiments, the glycolipid is provided an isolated form. In someembodiments, the glycolipid is provided in a micelle form or a liposomeform, alone or optionally with other agents such as antigens,polysaccharides and the like. In some embodiments, the glycolipid isprovided in a lyophilized form. Lyophilized forms glycolipid areparticularly suitable for long-term storage, ranging from days, weeks,months or even years. In some embodiments, the glycolipid is provided ina form that is suitable for administration to a human, including anorally administered form or a parenterally administered form.

In some embodiments, the glycolipid is obtained or derived from B.fragilis cells, including B. fragilis cells that overexpress PSArelative to polysaccharide B (PSB), PSC. PSD, PSE. PSF, PSG and PSH. Asused herein, obtained or derived intends that the glycolipid that isdefined structurally herein is rendered using the isolation methodsprovided herein. It does not however intend that any naturally occurringcounterpart of the glycolipid is structurally identical to the structuredefined herein.

In some embodiments, at least one acyl chain is unmodified. In someembodiments, at least one acyl chain is modified, for example modifiedwith a hydroxyl group. In some embodiments, at least one acyl chain isunmodified and at least one of the acyl chains is modified, for examplemodified with a hydroxyl group. In some embodiments, at least one acylchain is modified with a hydroxyl group. In some embodiments, at leastone acyl chain is C16:0-OH. In some embodiments, at least one acyl chainis C17:0-OH. In some embodiments, at least one acyl chain is C14:0. Insome embodiments, at least one acyl chain is C15:0.

In some embodiments, at least one acyl chain is N-substituted. In someembodiments, at least one acyl chain is O-substituted. In someembodiments, at least one acyl chain is N-substituted and at least oneacyl chain is O-substituted. In some embodiments, all of the acyl chainsare N-substituted or all of the acyl chains are O-substituted.

In some embodiments, the diglucosamine is phosphorylated. In someembodiments, the diglucosamine is mono phosphorylated. In someembodiments, the diglucosamine is not phosphorylated.

In some embodiments, the diglucosamine is conjugated to theoligosaccharide by an acid-labile bond. In some embodiments, theacid-labile bond is a ketosidic bond.

In some embodiments, the oligosaccharide comprises or contains 3-10sugars (e.g., it may comprise 3 or more sugars). The oligosaccharidehowever is not the polysaccharide of PSA (i.e., it is not thezwitterionic tetrasaccharide moiety or repeated moiety shown in FIG. 1). In some embodiments, the oligosaccharide contains 2-10, 2-9, 3-9,4-9, 5-9, 6-9, 7-9 or 8-9 sugars. In some embodiments, theoligosaccharide contains 6-9 sugars. In some embodiments, theoligosaccharide comprises galactose and/or glucose and/or fucoseresidues. Thus, the sugars of the oligosaccharide may be identical ordifferent from each other.

In some embodiments, the oligosaccharide comprises one or two KDOresidues. The oligosaccharide is not and does not comprise the repeatingtetrasaccharide units of PSA.

In some embodiments, the glycolipid is formulated with a detergent or abile salt. In some embodiments, the detergent or bile salt is present ator less than 1%, 0.5% or 0.1% (weight of detergent or bile salt/weightof glycolipid).

In some embodiments, the glycolipid is formulated with an antigen otherthan a lipidated PSA or a component of such lipidated PSA.

In some embodiments, the glycolipid is formulated with vaccineadditives.

In some embodiments, the glycolipid is formulated with a non-naturallyoccurring preservative. In some embodiments, the glycolipid isformulated with a non-naturally occurring stabilizer. In someembodiments, the glycolipid is formulated with human albumin, phenol,glycerin or glycine.

In some embodiments, the glycolipid is formulated with thimerosal,aluminum hydroxide, benzethonium chloride, formaldehyde, formalin,glutaraldehyde, potassium phosphate, aluminum potassium sulfate, bovineextract, calf serum, ammonium sulfate, aluminum phosphate, non-humancells. Vero (monkey kidney) cells, human cells, MRC-5 (human diploid)cells. MRC-5 cellular proteins, and the like.

In some embodiments, the glycolipid is formulated for parenteraladministration. In some embodiments, the glycolipid is formulated fororal administration.

Thus, also provided herein are compositions comprising any of theforegoing glycolipids. In some embodiments, the composition furthercomprises an antigen other than a lipidated PSA or a component of suchlipidated PSA. In some embodiments, the composition comprises any of theforegoing glycolipids and lipidated PSA in non-naturally occurringweight/weight ratios such as but not limited to 1:75, 1:50, 1:25, 1:20,1:10, 1:5, and the like.

In some embodiments, the composition further comprises a detergent or abile salt. In some embodiments, the detergent or bile salt is present ator less than 1%, 0.5% or 0.1% (weight of detergent or bile salt/weightof glycolipid).

In some embodiments, the composition further comprises vaccineadditives.

In some embodiments, the composition further comprises a non-naturallyoccurring preservative. In some embodiments, the composition furthercomprises a non-naturally occurring stabilizer. In some embodiments, thecomposition further comprises human albumin, phenol, glycerin orglycine.

In some embodiments, the composition further comprises thimerosal,aluminum hydroxide, benzethonium chloride, formaldehyde, formalin,glutaraldehyde, potassium phosphate, aluminum potassium sulfate, bovineextract, calf serum, ammonium sulfate, aluminum phosphate, non-humancells, Vero (monkey kidney) cells, human cells, MRC-5 (human diploid)cells, MRC-5 cellular proteins, and the like.

In some embodiments, any of the foregoing glycolipids or compositions isformulated for parenteral administration. In some embodiments, any ofthe foregoing glycolipids or compositions is formulated for oraladministration.

Also provided herein is a composition comprising a lipidatedpolysaccharide A (PSA) and any of the foregoing glycolipids in a 4:1 to20:1 ratio (w/w).

Also provided herein is a composition comprising a lipidatedpolysaccharide A (PSA) and at least 0.5% (w/w) of any of the foregoingglycolipids.

In some embodiments, the composition further comprises apharmaceutically acceptable carrier.

In some embodiments, the composition further comprises another therapysuch as an additional active agent or it is formulated foradministration in combination with another therapy such as an additionalactive agent. In some embodiments, the other therapy is or comprises acancer immunotherapy. In some embodiments, the cancer immunotherapycomprises administration of an antibody or antibody fragment thereof.

In some embodiments, the composition is formulated for parenteraladministration. In some embodiments, the composition is formulated fororal administration.

Also provided herein is a composition comprising any one of theforegoing glycolipids and an antigen. In some embodiments, thecomposition is a vaccine.

In some embodiments, the antigen is a bacterial antigen. In someembodiments, the antigen is attenuated bacteria. In some embodiments,the antigen is a viral antigen. In some embodiments, the antigen isattenuated virus. In some embodiments, the antigen is a fungal antigen.In some embodiments, the antigen is a mycobacterial antigen. In someembodiments, the antigen is a parasitic antigen. In some embodiments,the antigen is a cancer antigen. In some embodiments, the antigen is ahuman protein. In some embodiments, the antigen is a humanpolysaccharide. In some embodiments, the composition is formulated forparenteral administration.

Also provided herein is a method of enhancing an innate immune responsecomprising administering, to a subject in need of an immune response,such as an enhanced immune response, and further such as an enhancedinnate immune response, an effective amount of any of the foregoingglycolipids.

In some embodiments, the method further comprises administering alipidated PSA in combination with another therapy. In some embodiments,the other therapy is or comprises a cancer immunotherapy. In someembodiments, the cancer immunotherapy comprises administration of anantibody or antibody fragment thereof.

Also provided herein is a method of inducing an immune responsecomprising administering, to a subject in need of immune responseinduction or modulation, an effective amount of any of the foregoingglycolipids.

In some embodiments, the method further comprises administering alipidated PSA in combination with another therapy. In some embodiments,the other therapy is or comprises a cancer immunotherapy. In someembodiments, the cancer immunotherapy comprises administration of anantibody or antibody fragment thereof.

In some embodiments, the immune response is an innate immune response.In some embodiments, the immune response is an adaptive immune response.

Also provided herein is a method of inducing an immune responsecomprising administering, to a subject in need of immune responseinduction or modulation, an effective amount of any of the foregoingglycolipids, wherein the immune response comprises induction ofinterferon-beta, and the subject is experiencing or is likely toexperience an interferon-beta responsive condition (e.g., a conditionthat has been shown to benefit from administration of exogenousinterferon-beta and/or that can benefit from induction ofinterferon-beta in vivo). Such conditions include but are not limited toany of the autoimmune diseases provided herein. In some embodiments, thecondition is multiple sclerosis (MS). In some embodiments, the conditionis systemic lupus erythematosus.

Also provided herein is a method of inducing an immune response to anantigen comprising administering, to a subject in need of anantigen-specific immune response, an effective amount of any of theforegoing glycolipid and the antigen.

In some embodiments, the subject has or is at risk of developing abacterial infection. In some embodiments, the antigen is a bacterialantigen. In some embodiments, the antigen is attenuated bacteria.

In some embodiments, the subject has or is at risk of developing a viralinfection. In some embodiments, the antigen is a viral antigen. In someembodiments, the antigen is attenuated virus.

In some embodiments, the subject has or is at risk of developing afungal infection. In some embodiments, the antigen is a fungal antigen.

In some embodiments, the subject has or is at risk of developing amycobacterial infection. In some embodiments, the antigen is amycobacterial antigen.

In some embodiments, the subject has or is at risk of developing aparasitic infection.

In some embodiments, the antigen is a parasitic antigen.

In some embodiments, the subject has or is at risk of developing acancer. In some embodiments, the antigen is a cancer antigen.

In some embodiments, the antigen is a human protein. In someembodiments, the antigen is a human polysaccharide.

In some embodiments, the method further comprises administering to thesubject lipidated PSA.

Also provided herein is a micelle consisting essentially of any one orany combination of the foregoing glycolipids.

Also provided herein is a composition comprising a micelle consistingessentially of any one or any combination of the foregoing glycolipidsand a detergent or bile salt. In some embodiments, the detergent or bilesalt is present in a pharmaceutically acceptable amount. In someembodiments, the composition is a pharmaceutical composition. In someembodiments, formulated for parenteral administration.

In various embodiments, the glycolipid is free of non-lipidated PSA. Invarious embodiments, the glycolipid is free of lipidated PSA.

In various embodiments, the glycolipid is suitable for administration toa human.

In various embodiments, the composition is formulated for parenteral orenteral or oral administration to a subject. In various embodiments, thecomposition is formulated for lipophilic delivery, including for examplein a liposome or in an oil-based delivery system. The variouscompositions provided herein may be formulated as a capsule or otherdiscrete dosage form, including those intended for oral or enteraladministration.

In another aspect, the invention provides an isolated glycolipidcomprising a diglucosamine covalently conjugated to 3-5 acyl chains,each independently ranging in length from 14-17 carbons. The glycolipidmay be any of the glycolipids provided herein, or a combination thereof.In various embodiments, the diglucosamine is covalently conjugated to3-5 acyl chains. In various embodiments, the diglucosamine is covalentlyconjugated to 4 or 5 acyl chains. In various embodiments, the acylchains range in length from 15-17 carbons. Other embodiments relating tothe glycolipids are recited above and apply equally. It is to beunderstood that any of the aspects and embodiments described hereinrelating to glycolipids, including compositions and methods of use,embrace and contemplate glycolipids that comprise, consist or consistessentially of a disaccharide, such as a diglucosamine, covalentlyconjugated to 3-5 acyl chains, and optionally each chain mayindependently range in length from 4-17 carbons.

In another aspect, the invention provides a method comprisingadministering to a subject in need of immune response modulation acomposition comprising a lipidated polysaccharide A (PSA) and any of theforegoing glycolipids in an effective amount.

In another aspect, the invention provides a method comprisingadministering to a subject in need of immune response modulation aneffective amount of a composition comprising a lipidated polysaccharideA (PSA) and any of the foregoing glycolipids, in combination with anadditional therapy.

In another aspect, the invention provides a method comprisingadministering to a subject in need of immune response modulation one orboth of a composition comprising a lipidated polysaccharide A (PSA) andany of the foregoing glycolipids; and another therapy, so that thesubject receives therapy with the composition and the other therapy incombination.

In some embodiments, the subject has or is at risk of developing cancer.In some embodiments, the subject is diagnosed as suffering from,susceptible to, and/or receiving therapy for cancer.

In some embodiments, the additional therapy is or comprises a cancerimmunotherapy. In some embodiments, the cancer immunotherapy is orcomprises an antibody or antibody fragment thereof.

In some embodiments, the step of administering comprises administeringaccording to a regimen that has been demonstrated to achievestatistically significant improvement in immune function whenadministered to a relevant population of subjects. The relevantpopulation of subjects may comprise subjects diagnosed as sufferingfrom, susceptible to, and/or receiving therapy for cancer. In someembodiments, the improvement in immune function comprises modulation ofan innate and/or adaptive immune response.

In some embodiments, the subject in need of immune response modulationis a subject in need of innate immune response modulation. In someembodiments, the subject in need of immune response modulation is asubject in need of adaptive immune response modulation. In someembodiments, the subject in need of immune response modulation is asubject in need of immune response stimulation.

In some embodiments, improvement in immune function comprisesstimulation of an innate and/or adaptive immune response.

In some embodiments, the step of administering comprises administeringan amount of the composition effective to achieve an improvement inimmune function as compared to a subject who has not been administeredthe composition.

In some aspects, the acylated diglucosamine, such as but not limited tothe tetra-acylated diglucosamine or the penta-acylated diglucosamine,may be administered to a subject experiencing or likely to experience aproinflammatory immune response, such as may occur for example during anE. coli infection or an infection by gram negative bacteria. Theacylated diglucosamine may be conjugated to an oligosaccharide such as3-10-mer oligosaccharide. The subject may be defined as one experiencingor likely to experience a gram negative bacterial infection, includingbut not limited to infection by any of the gram negative bacterialspecies provided herein.

It is to be understood that various foregoing aspects and embodimentsoverlap. It is intended that the embodiments recited above apply equallyto the various aspects recited above.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein. It should also be appreciated that terminologyexplicitly employed herein that also may appear in any disclosureincorporated by reference should be accorded a meaning most consistentwith the particular concepts disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

It is to be understood that the Figures are not necessarily to scale,emphasis instead being placed upon generally illustrating the variousconcepts discussed herein.

FIG. 1 is a proposed generic structure of isolated B. fragilis lipidatedPSA. The structure includes a repeating tetrasaccharide unit having anaverage molecular weight of about 130 KDa, an oligosaccharide core unitcomprising on average less 10 or fewer sugar residues, and a glycolipid.The glycolipid may comprise 3-5 acyl chains that may be N or O linked toa diglucosamine. The diglucosamine may be mono phosphorylated ornon-phosphorylated.

FIG. 2 is one embodiment of a proposed generic structure of isolated B.fragilis glycolipid antigen (referred to herein as GLA) with norepeating tetrasaccharide attached. The structure includes a coreoligosaccharide unit attached to a diglucosamine-comprising glycolipidmoiety having 5 acyl chains. One glucosamine is phosphorylated. Thelinkage between the diglucosamine and the core oligosaccharide unit isan acid-labile ketosidic linkage. When the oligosaccharide core unit islinked to the repeating tetrasaccharide units of PSA (not shown), thisoccurs through a glycosidic linkage. The oligosaccharide core unit maycomprise galactose, glucose or fucose sugars. It may further contain KDOresidues that connect the diglucosamine to the remaining sugars of thecore unit. As illustrated, it comprises 6 galactose units conjugated to2 KDO residues conjugated, through a ketosidic linkage, to adiglucosamine conjugated to 5 acyl chains. The diglucosamine is monophosphorylated. As taught herein, though, the glycolipid may benon-phosphorylated, the diglucosamine may be tri- or tetra-acylated, andthere may be variation in the oligosaccharide core unit in terms ofnumber and nature of sugars. Accordingly. GLA may minimally comprise thediglucosamine linked to a plurality of acyl chains (e.g., 3-5 chains).

FIG. 3 is a bar graph showing that B. fragilis GLA activates TLR2similar to lipidated PSA. The graph further shows that GLA can activateTLR2 at much lower concentrations as compared to lipidated PSA. Thegraph shows IL-8 production in HEK cells that are stably transfectedwith TLR2 only (while, or first bar of each pair of bars) or transfectedwith TLR2 and TLR1 (diagonal striped, or second bar of each pair ofbars) in response to purified lipidated PSA that does not contain anyfree lipid (referred to as PSA39) and B. fragilis GLA alone. HEK cellsover-expressing TLR2 and TLR2/l were stimulated with PSA39 or B. fragGLA for 24 hours and IL-8 levels in the culture supernatant weremeasured with ELISA. The Figure shows that far less (e.g., about 20 foldless to about 100 fold less) GLA preparation is needed to stimulate thesame level of IL-8 expression from the H11EK cells. For example, the 100ng/ml of GLA induces about 100-fold more IL-8 than does the 10microgram/ml of PSA39, even though the latter dose contains about 100nanograms of GLA. TLR2 activation plays a role in innate immunity, sothe ability of GLA to stimulate TLR2 indicates that GLA is able tostimulate innate immunity. The ability of lipidated PSA (PSA39) tostimulate TLR2 may be due to its glycolipid moiety which comprises onlyabout 1% of the mass of the lipidated PSA.

FIGS. 4A-4C show that PSA with higher levels of GLA provides betterprotection in an EAE model than PSA alone. The Figures show that apreparation comprising lipidated PSA and GLA (at an approximately 4:1ratio, w/w, referred to herein as Lot 35L) protects in an EAE modelsystem better than purified lipidated PSA lacking any free glycolipid(Lot 39). C57BLU6 mice were orally administered purified lipidated PSA(Lot 39), or a preparation comprising lipidated PSA and “free” GLA (Lot35L), or PBS every other day starting from one week before inducing thedisease. Mice were challenged subcutaneously with MOG 35-55 in CompleteFreund's Adjuvant. On days 0 and 2 after challenge, the mice receivedintraperitoneal Bordetella pertussis toxin. Mice were monitored andscored daily for disease progression. FIG. 4A shows the clinical EAEscore of the mice, with a score of 5 indicating death. FIG. 4B shows the% weight loss of the mice. Significance of the results using Lot 35L toLot 39 or to PBS is indicated by white asterisks (Lot 39 (8/8) and greyasterisks (PBS (8/8)), respectively. FIG. 4C shows the percent survivalof the mice. Lot 35L, which contains more free GLA as compared to Lot39, reduced the severity of the disease and the cumulative diseasescore, and increased survival rate of the mice. The presence of free GLAenhanced the protective activity of lipidated PSA.

FIG. 5 provides various metrics using the same EAE mouse model asdescribed for FIGS. 4A-4C to show that PSA with higher levels of GLAprovides better protection in the EAE model than PSA alone. Thestatistical significance of the three parameters in the top row and theparameter in lower left were all p<0.01. For example, the upper leftplot shows the cumulative disease scores calculated as the sum of allEAE clinical scores for each group of mice. Purified lipidated PSA isindicated as 39 and a mixture of purified lipidated PSA and free GLA, ina roughly 4:1 w/w mixture, is indicated as 35L. In these experiments,about 100 micrograms of lipidated PSA (39) were used per mouse.Similarly about 100 micrograms of the 35L preparation were used permouse, corresponding to about 80 micrograms of lipidated PSA and 20micrograms of GLA.

FIGS. 6A-6D show that GLA acts as an adjuvant for lipidated PSA. Theexperiment was performed as explained for FIGS. 4A-4C. Lot 39 refers topurified lipidated PSA. GLA alone refers to purified GLA. Lot 39 and GLrefers to a preparation made by combining Lot 39 and GLA in roughly a4:1 w/w ratio. Similar results were obtained using a ratio of about100:1. FIG. 6A shows the clinical EAE score of mice subjected toMOG-peptide induced EAE (p<0.01). Significance of the results using Lot39 and GL together compared to GLA alone or Lot 39 alone is indicated byasterisks and circled asterisks (Lot 39), respectively, in FIG. 6A. FIG.6B shows the % weight loss in the mice (p<0.05). FIG. 6C shows thepercent survival of the mice. GLA alone failed to confer protection,while a reconstituted mixture of purified lipidated PSA and GLA (at a4:1 w/w ratio) protected animals from EAE. FIG. 6D shows the cumulativedisease scores calculated as the sum of all EAE clinical scores for eachgroup.

FIGS. 7A-7B show that a delipidated PSA preparation (referred to asPSA35 delip) cannot protect in EAE. PSA35 refers to a preparation thatcomprises lipidated PSA and free GLA. PSA35 delip refers to apreparation of PSA35 that has been acid hydrolyzed to cleave the lipidmoiety from lipidated PSA and has also undergone a lipid extraction withorganic solvent chloroform in order to remove all free lipid includingall released lipid. PSA35 delip likely comprises the repeatingtetrasaccharide polysaccharide chain of PSA conjugated to theoligosaccharide core unit. It lacks the diglucosamine-containingglycolipid. The experiment was performed as explained in FIGS. 4A-4C.FIG. 7A shows the clinical EAE score of mice subjected to MOG-peptideinduced EAE (p=0.001 on day 15). FIG. 7B shows the cumulative scorescalculated as the sum of all EAE clinical scores for each group.Delipidated PSA35 failed to confer protection, while PSA35 protectedanimals from EAE indicating the importance of the glycolipid in suchprotection. In these experiments, on the order of about 10 micrograms ofthe PSA35 preparation was administered per mouse, representing about 8micrograms of lipidated PSA and about 2 micrograms of GLA. This amountwas about 10-fold lower than the amounts used in the experiments of thepreceding Figures.

FIG. 8 is a schematic of a model of lipidated PSA inducedimmunomodulation. In this model, free GLA are shown to bind to a TLR2/1dimer via the acyl chains, thereby stimulating an innate immuneresponse. Although not shown, the free GLA may comprise theoligosaccharide core unit.

FIG. 9 shows the lipid profile of various lots of PSA (PSA39. PSA40 andPSA41) and GLA (GLA acap which was derived from an unencapsulated mutantand GLA41). Each lot was tested for the presence of tri-acylated,tri-acylated and phosphorylated, tetra-acylated, tetra-acylated andphosphorylated, penta-acylated, and penta-acylated and phosphorylatedglycolipid moieties. The bars represent the amount of each glycolipidmoiety as a percentage of the total lipid (y-axis). It is clear thateach preparation contains the same lipid species but in differentpercentages. In particular, PSA41 and GLA41 contain the same lipidspecies, although the lipids do not appear to be similarly distributedbetween the PSA and GLA lots.

FIG. 10 shows an in vitro dose response (IL-10 production) ofSpDC+Tcells co-cultured with dendritic cells and exposed to 10, 50 and250 mg/ml of PSA39, PSA40 and PSA41. PSA41 appears more potent in itsability to induce IL-10 as compared to other two preparations.

FIG. 11 shows that interferon-β is induced by B. fragilis glycolipidwhether or not attached to PSA. Model antigen presenting cells in theform of bone marrow derived dendritic cells (BMDCs) were stimulated for6 hours with B. fragilis polysaccharide A (PSA) or the purifiedglycolipid anchor (GLA unencapsulated mutant), followed by collection ofsupernatants. A cytokine ELISA was used to detect levels of interferon-β(IFNβ) in the supernatants. PSA doses are shown stoichiometrically. Thetwo curves would be superimposed if plotted on a molar basis because GLAis about 1% of a PSA molecule (i.e., 100 micrograms of PSA contain about1 microgram of GLA).

FIG. 12 shows that different acylation structures of B. fragilis GLAinduce low levels of pro-inflammatory cytokines such as TNFα. Modelantigen presenting cells in the form of bone marrow derived dendriticcells (BMDCs) were stimulated for 6 hours with tri-, tetra- orpenta-acylated B. fragilis GLA or hexa-acylated E. coli lipid A,followed by collection of supernatants. A cytokine ELISA was used todetect levels of TNFα in the supernatants. The three GLA species differin their ability to induce TNFα, but they all induce a much lower levelof TNFα as compared to E. coli lipid A.

FIG. 13 shows that tetra-acylated and penta-acylated B. fragilis lipid Aantagonize production of pro-inflammatory TNFα secretion by E. colihexa-acylated lipid A. Model antigen presenting cells in the form ofbone marrow derived dendritic cells (BMDCs) were stimulated for 6 hourswith purified tetra-acylated B. fragilis GLA and hexa-acylated E. colilipid A (in a 5:1 ratio) or with purified penta-acylated B. fragilis GLAand hexa-acylated E. coli lipid A (in a 5:1 ratio), followed bycollection of supernatants. A cytokine ELISA was used to detect levelsof TNFα in the supernatants. Both the tetra-acylated and penta-acylatedGLA were able to suppress the pro-inflammatory response to lipid A. Thissuggests a suppressive activity that may be useful, among other things,in treating gram negative bacterial infection.

DETAILED DESCRIPTION

Provided herein are compositions and methods of use relating to aglycolipid antigen comprising a diglucosamine-containing glycolipid. Theglycolipid antigen, referred to herein as GLA, minimally comprises adiglucosamine-containing glycolipid. The diglucosamine moiety may bemono phosphorylated or non-phosphorylated (e.g., dephosphorylated). Theglycolipid may comprise 3-5 acyl chains that are N- or O-linked to thediglucosamine.

The glycolipid may further be conjugated to an oligosaccharide, referredto herein as an oligosaccharide core unit. Such oligosaccharide maycomprise 10 or fewer sugars. The sugars may be galactose, glucose and/orfucose. The oligosaccharides are typically conjugated to thediglucosamine via an acid-labile linkage such as but not limited to aketosidic linkage.

Significantly, it has now been discovered, surprisingly, that theglycolipid is able to stimulate an innate immune response. This activityhas been heretofore unrecognized. The finding is significant since itindicates that lipidated PSA is able to stimulate both innate andadaptive immune responses via different portions of its structure. Evenmore surprising is that free glycolipid, when combined with lipidatedPSA, leads to enhanced protective activity in EAE mouse models. Thissuggests that free glycolipid is able to enhance immune responses to agreater degree than possible when used in its conjugated form. Thisdisclosure provides methods of use of this glycolipid as an adjuvanttogether with PSA, whether or not lipidated, as well as a variety ofother antigens. This newly discovered adjuvant activity indicates a roleof the glycolipid in enhancing innate immunity, including when usedalone, as well as in enhancing adaptive immunity when used together withan antigen. Examples of this latter application include use as anadjuvant for vaccines, and well as in immuno-oncology.

The glycolipid has been obtained from manipulation of B. fragilis cells.The starting material was a non-naturally, mutant occurring strain of B.fragilis that overexpresses PSA but not other capsular polysaccharides.The purification protocol may include a phenol/water extraction, andDNAse. RNase and pronase treatments. If the material is then treatedwith deoxycholate (DOC), the glycolipid comprising the oligosaccharidecore unit is obtained. DOC treatment therefore separates the glycolipidfrom lipidated PSA. If however the material is treated instead with acidhydrolysis, the acid-labile linkage between the oligosaccharide coreunit and the acyl-substituted diglucosamine is cleaved and the freeacyl-substituted diglucosamine is obtained. If instead a purification isperformed that does not involve either DOC treatment or acid hydrolysis(e.g., column chromatography), then the resulting preparation comprisesabout 20% (w/w) free glycolipid and about 80% (w/w) PSA includinglipidated PSA. One such preparation is used in the experiments describedherein, and is referred to as 35L or PSA35L or Lot 35L. As used herein,a “free” glycolipid refers to a glycolipid that is not covalentlyattached to a tetrasaccharide unit of PSA (i.e., it does not comprisethe polysaccharide portion of PSA that is comprised of repeatingtetrasaccharide units).

Structural analysis showed this lipid to belong to the class of lipid Amolecules normally found in Gram negative bacteria. GLA, as it isreferred to in this disclosure, comprises a glucosamine disaccharidewith 3 to 5 acyl chains covalently bound to the sugars. It can be a monophosphorylated or non-phosphorylated at the diglucosamine. One of theglucosamine residues is linked to an oligosaccharide core of 10 or fewersugars, including 9, 8, 7 or less sugars, by an acid labile bond such asa ketosidic bond.

In order to enhance the harvest of intact glycolipid, the acidhydrolysis step was eliminated and lots were prepared with lipidated PSAand the free glycolipid. Such preparations are identified herein as 35Lor PSA35L. These preparations were further analyzed and found tocomprise on average about a 4:1 ratio of lipidated PSA to free GLA(w/w). In the in vivo EAE model, treatment with the 35L preparationdelayed the EAE clinical outcome, and reduced the severity and thecumulative scores of the disease as compared to a purified lipidated PSApreparation referred to herein as PSA39 (DOC treated lipidated PSApreparation that did not contain free lipid (FIG. 4A, FIG. 5 upper leftcorner, and FIG. 4C). Next a mixture of free lipid and lipidated PSA wasreconstituted at a 4:1 ratio (w/w), and this mixture was tested in thesame experiment. The reconstituted mixture, referred to herein as “Lot39+GL” conferred protection similar to that seen with 35L (FIGS. 6A, 6Cand 6D). Moreover, when the PSA35L preparation (comprising lipidated PSAand free lipid in a 4:1 w/w ratio) was hydrolyzed and delipidated afterpurification, it lost its ability to protect (FIGS. 7A-7B).

Further analysis revealed that GLA activates TLR2/TLR 1 expressing HEKcells in a fashion similar to that seen with lipidated PSA (FIG. 3 ).

Taken together, the data suggest that GLA acts as an adjuvant to induceinnate immunity. Such activity may occur through engagement andsignaling of the TLR2/TLR1 heterodimer complex, and it likely enhancesimmune responses to PSA. In addition, the findings indicate use of GLAin enhancing immune responses to other antigens including but notlimited to bacterial antigens, cancer antigens, and the like.

Provided herein is the structural identification and characterization ofthe glycolipid moiety of lipidated PSA. It has been found in accordancewith this disclosure that these glycolipids are comprised of adiglucosamine substituted with three, four or five acyl chains. Theglycolipid in some instances further comprises an oligosaccharide coreunit conjugated to the diglucosamine via an acid-labile ketosidic bond.This glycolipid has been shown to activate an innate immune response andto enhance an immune response to lipidated PSA.

The glycolipid structure will now be described in greater detail in thecontext of lipidated PSA.

Lipidated PSA

A form of lipidated PSA isolated from mutant B. fragilis cells comprisesa polysaccharide component conjugated, at its reducing end, anoligosaccharide core unit, and a diglucosamine conjugated to 3-5 acylchains. As described in greater detail herein, the innateimmunostimulatory activity appears attributable to the diglucosaminesubstituted with acyl chains. The oligosaccharide core unit, by virtueof its hydrophilicity, helps to render the lipid component more watersoluble. Thus, while the immunostimulatory activity is believed to existin the acyl chains, typically the GLA of this disclosure will furthercomprise the oligosaccharide core unit in order to facilitate itsdissolution in an aqueous solution, such as but not limited to apharmaceutical formulation.

Polysaccharide Component

The polysaccharide component of lipidated PSA, referred to herein asPSA, comprises a tetrasaccharide repeating unit shown below. Itpossesses zwitterionic behavior as conferred by a positive charge on itsfree amine group and a negative charge on its free carboxyl group (perrepeating tetrasaccharide unit). Its naturally occurring state has beenreported to comprise over 60 tetrasaccharide repeating units (e.g., upto and including in some instances about 100, or about 200, or about 300repeated units on average), and it has an average molecular size ofabout 150 kD (with a range of about 75 kD to 240 kD).

The repeating tetrasaccharide unit of PSA has a structure as follows:

The tetrasaccharide repeating unit may also be expressed as follows:

Synthetic forms of lipidated PSA may comprise comprising various rangesof tetrasaccharide units (e.g., 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9,1-8, 1-7, 1-6, or 1-5 tetrasaccharide units). Such shorter variants canbe obtained by depolymerizing naturally occurring lipidated PSA or bydepolymerizing PSA obtained from lipidated PSA. PSA can be depolymerizedusing for example chemical means (e.g., using reactive oxygen species orreactive nitrogen species such as but not limited to nitrogen monoxide,as described in Duan and Kasper, Glycobiology, 2011, 21(4):401-409),mechanical means, and/or enzymatic means that are known in the art.

The invention further contemplates synthetic forms of lipidated PSAcomprising more than 300 repeating tetrasaccharide units, includingwithout limitation 350, 400, 500, 600, 700, 800, 900 or 1000 units ormore.

As described herein, the polysaccharide component may be covalentlyconjugated to the glycolipid, or in certain synthetic forms it may beunconjugated to the glycolipid. If covalently conjugated, it may beconjugated via a glycosidic bond to the oligosaccharide core unit. Inother embodiments, it may be conjugated via a ketosidic bond or otheracid labile bond or via a bond such as an ester, an amide, or an etherbond to form a non-naturally occurring lipidated PSA.

Glycolipid Component

The glycolipid component comprises a diglucosamine substituted with oneor more acyl chains, and preferably 3, 4 or 5 acyl chains. An exemplarydiglucosamine in the context of a glycolipid is provided in FIG. 2 . Itis now recognized in accordance with this disclosure that thediglucosamine is conjugated to the oligosaccharide core unit via aketosidic bond that is acid-labile and thus susceptible to stringenthydrolysis.

The disaccharide may be conjugated to one or more acyl chains, includingthree, four, or five acyl chains in some instances via for example esteror amide linkages, and thus may be referred to as “O” substituted (orO-linked) or “N” substituted (or N-linked) respectively. In someinstances, each glycolipid (or GLA, as used herein) comprises three,four or five acyl chains. Accordingly, the glycolipid may be referred toherein as tri-acylated, tetra-acylated or penta-acylated forms.

The acyl chains may range in length from 14 to 17 carbons, in someinstances. The acyl chains may be unmodified or they may be modified. Ifmodified, the acyl chains may be hydroxy-modified. Thus, in someinstances, the glycolipid may comprise one or more acyl chainscharacterized as C14:0, C14:0-OH, C15:0, C15:0-OH, C16:0, C16:0-OH,C17:0, and C17:0-OH.

A single preparation of glycolipid may yield a number glycolipid speciesthat diffe from each other with respect to acyl chain number. The exactcomposition of a glycolipid may be determined using mass spectrometry(MS), wherein different glycolipid species give rise to different anddiscernable spectra.

Examples of different penta-acylated species include:

(1) one chain of C16:0-OH, three chains of C17:0-OH, and one chain ofC15:0,

(2) two chains of C16:0-OH, two chains of C17:0-OH, and one chain ofC15:0,

(3) three chains of C16:0 OH, one chain of C17:0-OH, and one chain ofC15:0,

(4) four chains of C16:0-OH, and one chain of C15:0, and

(5) four chains of C16:0-OH, and one chain of C14:0.

Various species of tetra-acylated and tri-acylated species are similarlycontemplated.

It will therefore be appreciated glycolipids of this disclosure, whetherisolated from B. fragilis or generated de novo, and whether conjugatedor unconjugated to an oligosaccharide core unit, may comprise any of theforegoing combinations of acyl chains, without limitation:

(1) C16:0-OH acyl chain(s) only,

(2) C17:0-OH acyl chain(s) only,

(3) C16:0-OH and C17:0-OH chain(s) only,

(4) C16:0-OH and C17:0-OH and C15:0 chain(s) only.

(5) C16:0-OH and C17:0-OH and C14:0 chain(s).

The number of each type of chain may vary, and may include withoutlimitation the following options

(1) 0-4 C16:0-OH chains,

(2) 0-4 C17:0-OH chains,

(3) 0 or 1 C14:0 chains, and

(4) 0 or 1 C15:0 chains.

The foregoing examples are not to be considered limiting, and rather theinvention contemplates various combinations, and combinations of theforegoing, to be used in compositions provided herein.

The invention provides defined glycolipid mixtures, having known, andthus optionally pre-defined, glycolipid content and composition, andoptionally known and/or pre-defined polysaccharide (e.g., PSA) toglycolipid ratios or non-PSA antigen to glycolipid ratios. Thus, theglycolipids of this disclosure may be characterized in terms of any ofthese structural features, thereby further distinguishing these productsfrom those of the prior art, and where necessary from naturallyoccurring products. For example, based on the teachings provided herein,the invention provides compositions comprising glycolipids that are onlyor predominantly (e.g., greater than 50%, or at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%) tri-acylated, or tetra-acylated, orpenta-acylated, or some combination thereof including but not limited totetra- and penta-acylated. Such chemically defined compositions were notheretofore contemplated or possible.

Isolated Forms

The preparations provided herein can be characterized by their contentof released or free glycolipids. Such content can be about 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less. Such content can be less than35%, less than 30%, less than 25%, less than 20%, less than 15%, lessthan 10%, less than 5%, less than 4%, less than 3%, less than 2%, lessthan 1%, less than 0.5%, less 0.1%, less than 0.05%, less than 0.001%,less than 0.0005%, less than 0.0001% (w/w of released glycolipid tolipidated PSA). The amount of glycolipid may be determined for exampleusing the gel electrophoresis methods or mass spec methods.

The glycolipid may also be considered to be pure (i.e., it is free orsubstantially free of contaminant). The degree of purity may be at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or higher.

Accordingly, the invention provides compositions comprising glycolipidsincluding compositions comprising isolated glycolipid at a purity and/ora concentration that has not been heretofore achieved. Also provided arecompositions comprising or consisting essentially of particular speciesof glycolipid or particular subsets of species of glycolipid. Thesespecies may be characterized and thus distinguished from other speciesand from bulk glycolipid in terms of their lipid and oligosaccharidecomponents. The lipid components may be characterized by the number,position and type of acyl chains they possess. For example, acomposition may comprise at least 70%, 75%, 80%, 85%, 90%, 95%, or momof a tri-acetylated, tetra-acylated and/or penta-acylated glycolipid.

The compositions may be defined by their degree of purity, for examplewith respect to their glycolipid components, or with respect to theircontent of contaminants such as non-lipidated PSA. The compositions maybe defined by their concentration of glycolipid components, or by theirratio of glycolipid to other components.

Synthetic Forms

The invention further provides additional synthetic, non-naturallyoccurring species of glycolipids.

Glycolipid Compositions

Isolated intends that the glycolipid (or other agent) is provided in amore pure form or a more concentration form compared to its synthesizedor obtained form. An “isolated” glycolipid may be a glycolipid that isprepared or obtained from B. fragilis, and is physically separated froma natural environment (e.g., a B. fragilis cell, components of the B.fragilis cell, and/or components of the B. fragilis cell capsularcomplex such as but not limited to PSB). It does not intend that anynaturally occurring product has the same structure of glycolipid asdescribed herein.

In some embodiments, the compositions are substantially free ofnaturally occurring contaminants such as nucleic acids (e.g., DNA andRNA), proteins, and other components of B. fragilis and/or the B.fragilis capsule. Substantially free, as used herein, intends that thesecontaminants represent about or less than 5%, less than 1%, less than0.5%, or less than 0.1% (or less) by weight (weight of the contaminantto weight of the glycolipid). In some instances, such contaminants maybe undetectable.

Various compositions may or may not contain LPS. LPS may be present inan amount of about 0.5% (w/w of LPS relative to other components in acomposition).

Some compositions may comprise at least about 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or more (w/w) of glycolipid.

Some compositions may comprise at least about 95%, 96%, 97%, 98%, 99%,or more (w/w) of lipidated PSA and less than 5%, less than 4%, less than3%, less than 2%, less than 1%, less than 0.5%, or less of free,released glycolipid.

Methods of Use

Also provided are methods of using the various forms of glycolipid invitro and in vivo. The various glycolipid forms herein can be used asimmunomodulators including immunostimulators, particularly in view oftheir enhanced ability to stimulate antigen presenting cells such asplasmacytoid dendritic cells. These forms are contemplated for use invitro and in vivo. In vitro uses include use as an analytical tool(e.g., for screening assays) and as an assay standard or control (e.g.,as a positive control or a comparator in an in vitro assay such as aIL-8 induction assay). In vivo uses include uses in animal models andalso in human and non-human subjects to treat or prevent conditions thatwould benefit from such immunomodulation including immunostimulation.Such conditions include those in which an enhanced innate and/oradaptive immune response is beneficial. Other conditions include but arenot limited to autoimmune disorders (e.g., multiple sclerosis andinflammatory bowel disease).

The glycolipid component may be used as a single agent, or it may beused in combination with other agents such as antigens or cells such asantigen-presenting cells. It may be used to stimulate antigen-presentingcells in vivo or in vitro. One application may involve obtainingantigen-presenting cells from a subject, stimulating such cells ex vivowith the glycolipid of this disclosure with or without antigen, followedby re-introduction of such cells with and without glycolipid and/orantigen to the subject. Alternatively, the glycolipid may beadministered to a subject with and/or without antigen. Such immunizationor vaccination protocols may involve several administrations ofglycolipid and/or antigen. The administration of the glycolipid and theantigen may occur simultaneously (typically occurs if they areformulated together), or substantially simultaneously (typically occursif they are formulated and administered separately but still within ashort period of time in order to work in concert in a subject), and/orthey may be formulated and administered separately (e.g., the glycolipidis administered before or after the antigen, including for examplehours, days and/or weeks before or after).

The invention further contemplates use of the free polysaccharidecomponent of lipidated PSA (i.e., a chain of repeating tetrasaccharideunits that is not conjugated to the glycolipid component of lipidatedPSA (including where such glycolipid comprises the oligosaccharide coreunit) and the free glycolipid component obtained from lipidated PSA.

As another example, the polysaccharide and glycolipid may be usedtogether in an unconjugated form.

The invention further contemplates use of the free PSA (i.e., a chain ofrepeating tetrasaccharide units that is not conjugated to the glycolipid(including where such glycolipid comprises the oligosaccharide coreunit) and free glycolipid obtained from lipidated PSA.

In vivo uses include but are not limited to those involving humansubjects. For example, in vivo uses include administration of theglycolipid and compositions thereof to a non-human subject in order tomodulate an immune response, for example as a positive control or acomparator.

Infections

The glycolipids and compositions thereof may be administered to asubject having or at risk of having an infection such as a bacterialinfection, a viral infection, a fungal infection, a parasitic infection,or a mycobacterial infection. In these aspects, the glycolipids may beused and/or administered with another therapeutic such as ananti-bacterial agent, an anti-viral agent, an anti-fungal agent, ananti-parasitic agent, or anti-mycobacterial agent. In these and otheraspects, the glycolipids may be used and/or administered with an antigenin order to induce or enhance an immune response directed to the antigen(i.e., an antigen-specific immune response). Such antigens includebacterial antigens, viral antigens, fungal antigens, parasitic antigens,and mycobacterial antigens.

Anti-bacterial agents include broad spectrum antibiotics, narrowspectrum antibiotics, or limited spectrum antibiotics. Theanti-bacterial agent may also be a cell wall synthesis inhibitor, cellmembrane inhibitor, protein synthesis inhibitor, nucleic acid synthesisor functional inhibitor or a competitive inhibitor.

Anti-viral agents include immunoglobulin, amantadine, interferon,nucleoside analogue, nonnucleoside analogue, bioflavonoid and proteaseinhibitor, although it is not so limited. In one embodiment, theprotease inhibitor is indinavir, saquinavir, ritonavir, and nelfinavir.In another embodiment, the bioflavonoid is robustaflavone,amentoflavone, or a derivative or salt thereof. In yet anotherembodiment, the non-nucleoside analogue is selected from the groupconsisting of delavirdine, nevirapine, efavirenz, alpha-interferon,recombinant CD4, amantadine, rimantadine, ribavirin and vidarabine.

Anti-fungal agents include imidazole. FK 463, amphotericin B. BAY38-9502, MK 991, pradimicin, UK 292, butenafine, chitinase and 501cream.

Anti-parasitic agents include albendazole, amphotericin B, benznidazole,bithionol, chloroquine HCl, chloroquine phosphate, clindamycin,dehydroemetine, diethylcarbamazine, diloxanide furoate, eflornithine,furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin,mebendazole, mefloquine, meglumine antimoniate, melarsoprol,metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine,paromomycin, pentamidine isethionate, piperazine, praziquantel,primaquine phosphate, proguanil, pyrantel pamoate,pyrimethamine-sulfonamides, pyrimethamine-sulfadoxine, quinacrine HCl,quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium(sodium antimony gluconate), suramin, tetracycline, doxycycline,thiabendazole, tinidazole, trimethroprim-sulfamethoxazole, andtryparsamide.

Anti-mycobacterial agent include anti-tuberculosis agent such asisoniazid, rifampin, rifabutin, rifapentine, pyrazinamide, ethambutol,(+)calanolide A, (−)-calanolide A, (−)-soulattrolide, (−)-costatolide or(−)-7,8-dihydrosoulattrolide. Other anti-mycobacterial agents includestreptomycin, dapsone, clarithromycin, ciprofloxacin, clofazamine,azithromycin, ethionamide, amikacin or resorcinomycin A.

Subjects to be treated according to this disclosure may have or may beat risk of developing a bacterial infection. Examples of bacterialinfections to be treated according to this disclosure include but arenot limited to an E. coli infection, a Staphylococcal infection, aStreptococcal infection, a Pseudomonas infection, Clostridium difficileinfection. Legionella infection, Pneumococcus infection, Haemophilusinfection, Klebsiella infection, Enterobacter infection, Citrobacterinfection, Neisseria infection, Shigella infection, Salmonellainfection, Listeria infection, Pasteurella infection, Streptobacillusinfection. Spirillum infection, Treponema infection, Actinomycesinfection, Borrelia infection, Corynebacterium infection, Nocardiainfection, Gardnerella infection, Campylobacter infection, Spirochaetainfection, Proteus infection, Bacteroides infection, H. pyloriinfection, and anthrax infection.

In some embodiments, the subjects may have or may be at risk ofdeveloping a gram-negative bacterial infection. Gram negative bacteriainclude but are not limited to Escherichia coli (E. coli), Pseudomonasaeruginosa, Neisseria meningitides, Neisseria gonorrhoeae, Chlamydiatrachomatis, Yersinia pestis, Non-typable Hemophilus influenzae,Hemophilus ducreyi, Helicobacter pylori, Campylobacter jejuni;Bacteroides fragilis. B. thetaiotamicron, B vulgatis, Citrobacterrodentium, Haemophilus influenza, Vibrio cholerae, Salmonella shigella,Salmonella enteritidis. Salmonella enterica serovar typhi. Salmonellaenterica serovar typhimurium, Klebsiella pneumoniae, Legionellapneumophila, Proteus mirabilis, Enterobacter cloacae, and Serratiamarcescens. In some embodiments, the tetra- and penta-acylatedglycolipids provided herein, and compositions thereof, are used to treatgram-negative bacterial infections, such as any of the foregoing.

In some embodiments, the subject is experiencing sepsis.

In some embodiments, the subject does not have and is not at risk ofdeveloping a B. fragilis infection. In some embodiments, the subjectdoes have or is at risk of developing a B. fragilis infection or acondition associated with type of infection.

Subjects to be treated according to this disclosure may have or may beat risk of developing a mycobacterial infection. Examples ofmycobacterial infections to be treated according to this disclosureinclude but arm not limited to tuberculosis or leprosy respectivelycaused by the M. tuberculosis and M. leprae species.

Subjects to be treated according to this disclosure may have or may beat risk of developing a viral infection. Examples of viral infections tobe treated according to this disclosure include but are not limited to aZika infection, an HIV infection, a Herpes simplex virus 1 infection, aHerpes simplex virus 2 infection, cytomegalovirus infection, hepatitis Avirus infection, hepatitis B virus infection, hepatitis C virusinfection, human papilloma virus infection, Epstein Barr virusinfection, rotavirus infection, adenovirus infection, influenza A virusinfection, respiratory syncytial virus infection, varicella-zoster virusinfections, small pox infection, monkey pox infection, and SARSinfection.

Subjects to be treated according to this disclosure may have or may beat risk of developing a parasitic infection. Examples of parasiticinfections to be treated according to this disclosure include but arenot limited to candidiasis, ringworm, histoplasmosis, blastomycosis,paracoccidioidomycosis, crytococcosis, aspergillosis, chromomycosis,mycetoma infections, pseudallescheriasis, and tinea versicolorinfection. Still other parasitic infections that may be treated usingthe glycolipids of this disclosure include amebiasis, Trypanosoma cruziinfection, Fascioliasis. Leishmaniasis, Plasmodium infections,Onchocerciasis. Paragonimiasis, Trypanosoma brucei infection,Pneumocystis infection, Trichomonas vaginalis infection. Taeniainfection, Hymenolepsis infection, Echinococcus infections,Schistosomiasis, neurocysticercosis, Necator americanus infection, andTrichuris trichiura infection.

Immune Therapies

The glycolipids and compositions thereof may be used to enhance immunetherapies for a number of indications, both in a therapeutic and aprophylactic sense. Immune therapies include but are not limited topassive immune therapies such as immunoglobulin administration, andactive immune therapies such as vaccination with antigens alone orantigens in the context of dendritic cells. The methods are intended totreat or prevent various indications that would benefit from an enhancedimmune response.

In some embodiments, the immune response that is stimulated is acell-mediated immune response involving antigen presenting cells andoptionally T cells such as Treg cells. In some embodiments, the immuneresponse is an innate immune response, while in others it is an adaptiveimmune response, while in yet others it is a combined innate andadaptive immune response. The immune response may, in some instances, beantigen specific.

The immune therapy may involve administration of an antibody orantigen-binding antibody fragment. The antibody or antigen-bindingfragment thereof may be specific for a tumor vasculature molecule. Tumorvasculature molecules include but are not limited to endoglin, ELAM-1,VCAM-1. ICAM-1, ligand reactive with LAM-1, MHC class H antigens, aminophospholipids such as phosphatidylserine and phosphatidylethanolamine,VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). Antibodies to endoglin includeTEC-4 and TEC-11. Antibodies that inhibit VEGF include 2C3 (ATCC PTA1595). Other antibodies that are specific for tumor vasculature includeantibodies that react to a complex of a growth factor and its receptorsuch as a complex of FGF and the FGFR or a complex of TGFβ and theTGFβR. Antibodies of this latter class include GV39 and GV97.

In a related embodiment, the antibody or antibody fragment is selectedfrom the group consisting of trastuzumab, alemtuzumab (B cell chroniclymphocytic leukemia), gemtuzumab ozogamicin (CD33+acute myeloidleukemia), hP67.6 (CD33+acute myeloid leukemia), infliximab(inflammatory bowel disease and rheumatoid arthritis), etanercept(rheumatoid arthritis), rituximab, tositumomab, MDX-210, oregovomab,anti-EGF receptor mAb, MDX-447, anti-tissue factor protein (TF),(Sunol); ior-c5, c5, edrecolomab, ibritumomab tiuxetan, anti-idiotypicmAb mimic of ganglioside GD3 epitope, anti-HLA-Dr10 mAb, anti-CD33humanized mAb, anti-CD52 humAb, anti-CD1 mAb (ior t6), MDX-22,celogovab, anti-17-1A mAb, bevacizumab, daclizumab, anti-TAG-72(MDX-220), anti-idiotypic mAb mimic of high molecular weightproteoglycan (I-Mel-1), anti-idiotypic mAb mimic of high molecularweight proteoglycan (I-Mel-2), anti-CEA Ab, hmAbH11, anti-DNA orDNA-associated proteins (histones) mAb. Gliomab-H mAb, GNT-250 mAb,anti-CD22, CMA 676), anti-idiotypic human mAb to GD2 ganglioside, ioregf/r3, anti-ior c2 glycoprotein mAb, ior c5, anti-FLK-2/FLT-3 mAb,anti-GD-2 bispecific mAb, antinuclear autoantibodies, anti-HLA-DR Ab,anti-CEA mAb, palivizumab, bevacizumab, alemtuzumab. BLyS-mAb,anti-VEGF2, anti-Trail receptor; B3 mAb, mAb BR96, breast cancer; andAbx-Cbl mAb.

The antibody or antibody fragment may be an anti-HER2 antibody, such astrastuzumab. The antibody or antibody fragment may be an anti-CD20antibody, such as rituximab.

In still another aspect, provided herein is a method for shortening avaccination course. As used herein, “shortening a vaccination course”refers to reducing either the number of vaccine administrations (e.g.,by injection) or the time between vaccine administrations. This isaccomplished by stimulating a more robust immune response in thesubject. The method may involve, in one embodiment, administering to asubject in need of immunization a glycolipid in an amount effective toinduce an antigen-specific immune response to a vaccine administered ina vaccination course, wherein the vaccination course is shortened by atleast one immunization. In other embodiments, the vaccination course isshortened by one immunization, two immunizations, three immunizations,or more. The method may involve, in another embodiment, administering toa subject in need of immunization a glycolipid of this disclosure in anamount effective to induce an antigen-specific immune response to avaccine administered in a vaccination course, wherein the vaccinationcourse is shortened by at least one day. In other embodiments, thevaccination course is shortened by one day, two days, three days, fourdays, five days, six days, one week, two weeks, three weeks, four weeks,one month, two months or more. In one embodiment, the glycolipid isadministered substantially simultaneously with the vaccine.

Immunizations that can be modified in this way include but are notlimited to newborn immunizations for HBV; immunizations at for exampletwo months of age for Polio. DTaP, Hib, HBV, Pneumococcus; immunizationsat for example four months of age for Polio, DTaP, Hib, Pneumococcus;immunizations at for example six months of age for Polio, DTaP, Hib,HBV, Pneumococcus; immunizations at for example 12-15 months of age forHib, Pneumococcus, MMR, Varicella; immunizations at for example 15-18months of age for DtaP; immunizations at for example 4-6 years of agefor Polio, DPT, MMR; immunizations at for example 11-12 years of age forMMR; immunizations at for example 14-16 years of age fortetanus-diphtheria (i.e., Td) (with a repeat as a booster every 10years).

As an example, a recommended vaccination course for tetanus/diphtheriaincludes a primary immunization series given in adults if not receivedas a child, followed by routine booster doses of tetanus-diphtheria (Td)every 10 years. The method of the invention will allow for a shortenedseries of vaccinations at the first time point, and may in someinstances obviate the need for booster shoots later on. As anotherexample, hepatitis vaccination commonly requires three administrationsspaced at least two weeks, and sometimes one month, apart in order todevelop full immunity. Using the methods of the invention, it ispossible to either reduce the number of injections from three to two orone, or to reduce the time in between injections from weeks or months todays or weeks. Vaccination courses that can be shortened by the methodof the invention include but are not limited to: HBV: Hepatitis Bvaccine (3 total doses currently recommended); Polio: Inactivated poliovaccine (4 total doses currently recommended); DTaP:Diphtheria/tetanus/acellular Pertussis (3-in-1 vaccine; 5 total dosescurrently recommended); Hib: Haemophilus influenzae type b conjugatevaccine (4 total doses currently recommended); Pneumococcus (Prevnar):Protects against certain forms of Strep. Pneumoniae (3 total dosesrecommended); MMR: measles/mumps/rubella (3-in-1 vaccine; 2 total dosesrecommended): Td: Adult tetanus/diphtheria (2-in-1 vaccine; for use inpeople over age 7). In another embodiment, the glycolipids can be usedtogether with oral polio vaccine.

Cancer

The glycolipids and compositions thereof may be administered to asubject having or at risk of developing cancer. The cancer may be onefor which a cancer antigen is known.

Examples of cancer that may be treated according to this disclosureinclude basal cell carcinoma, biliary tract cancer; bladder cancer: bonecancer, brain cancer, breast cancer, cervical cancer; choriocarcinoma;CNS cancer; colon and rectum cancer; connective tissue cancer; cancer ofthe digestive system; endometrial cancer; esophageal cancer; eye cancer,cancer of the head and neck; gastric cancer; intra-epithelial neoplasm;kidney cancer: larynx cancer; leukemia; acute myeloid leukemia, acutelymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia,liver cancer; small cell lung cancer; non-small cell lung cancer,lymphoma, Hodgkin's lymphoma; Non-Hodgkin's lymphoma; melanoma; myeloma;neuroblastoma; oral cavity cancer; ovarian cancer, pancreatic cancer;prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer, renalcancer; cancer of the respiratory system; sarcoma; skin cancer, stomachcancer; testicular cancer; thyroid cancer; uterine cancer; and cancer ofthe urinary system.

The glycolipids and compositions thereof may be administered withanother therapeutic agent or modality such as but not limited toantibody or other biologic, chemotherapy, radiation, surgery and thelike. The glycolipids and compositions thereof may be administered witha cancer antigen or a cancer immunotherapy such as an antibody specificfor a cancer antigen.

The chemotherapy may be selected from the group consisting ofaldesleukin, asparaginase, bleomycin sulfate, carboplatin, chlorambucil,cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, daunorubicin hydrochloride, docetaxel, doxorubicin,doxorubicin hydrochloride, epirubicin hydrochloride, etoposide,etoposide phosphate, floxuridine, fludarabine, fluorouracil,gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicinhydrochloride, ifosfamide, interferons, interferon-α2a, interferon-α2b,interferon-αn3, interferon-α1b, interleukins, irinotecan,mechlorethamine hydrochloride, melphalan, mercatopurine, methotrexate,methotrexate sodium, mitomycin, mitoxantrone, paclitaxel, pegaspargase,pentostatin, prednisone, profimer sodium, procabazine hydrochloride,taxol, taxotere, teniposide, topotecan hydrochloride, vinblastinesulfate, vincristine sulfate or vinorelbine tartrate.

The cancer immunotherapy may include an antibody or antibody fragmentspecific for a cell surface molecule. Cell surface molecules that may betargeted with the antibody or antibody fragment include but are notlimited to HER 2, CD20, CD33, EGF receptor, HLA markers such as HLA-DR,CD52, CD1, CEA, CD22, GD2 ganglioside, FLK2/FLT3, VEGF, VEGFR, and thelike.

The antibody or antibody fragment may be specific for a cancer antigen.Cancer antigens that may be targeted with the antibody or antibodyfragment include but are not limited to HER 2 (p185). CD20, CD33. GD3ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milkmucin core protein, TAG-72. Lewis A antigen, ovarian associated antigenssuch as OV-TL3 and MOv18, high Mr melanoma antigens recognized byantibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like. Othercancer antigens are described in U.S. Pat. No. 5,776,427.

Cancer antigens can be classified in a variety of ways. Cancer antigensinclude antigens encoded by genes that have undergone chromosomalalteration. Many of these antigens are found in lymphoma and leukemia.Even within this classification, antigens can be characterized as thosethat involve activation of quiescent genes. These include BCL-1 and IgH(Mantel cell lymphoma). BCL-2 and IgH (Follicular lymphoma), BCL-6(Diffuse large B-cell lymphoma). TAL-1 and TCRδ or SIL (T-cell acutelymphoblastic leukemia), c-MYC and IgH or IgL (Burkitt lymphoma),MUN/IRF4 and IgH (Myeloma), PAX-5 (BSAP) (Immunocytoma).

Other cancer antigens that involve chromosomal alteration and therebycreate a novel fusion gene and/or protein include RARα, PML, PLZF, NPMor NuMA (Acute promyelocytic leukemia), BCR and ABL (Chronicmyeloid/acute lymphoblastic leukemia), MLL (HRX) (Acute leukemia). E2Aand PBX or HLF (B-cell acute lymphoblastic leukemia). NPM, ALK(Anaplastic large cell leukemia), and NPM, MLF-1 (Myelodysplasticsyndrome/acute myeloid leukemia).

Other cancer antigens are specific to a tissue or cell lineage. Theseinclude cell surface proteins such as CD20, CD22 (Non-Hodgkin'slymphoma. B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD52(B-cell CLL), CD33 (acute myelogenous leukemia (AML)), CD10 (gp100)(common (pre-B) acute lymphocytic leukemia and malignant melanoma),CD3/T-cell receptor (TCR) (T-cell lymphoma and leukemia), CD79/B-cellreceptor (BCR) (B-cell lymphoma and leukemia). CD26 (epithelial andlymphoid malignancies), human leukocyte antigen (HLA)-DR. HLA-DP, andHLA-DQ (lymphoid malignancies), RCAS1 (gynecological carcinomas, bilaryadenocarcinomas and ductal adenocarcinomas of the pancreas), andprostate specific membrane antigen (prostate cancer).

Tissue- or lineage-specific cancer antigens also include epidermalgrowth factor receptors (high expression) such as EGFR (HER1 or erbB1)and EGFRvIII (brain, lung, breast, prostate and stomach cancer), erbB2(HER2 or HER2/neu) (breast cancer and gastric cancer), erbB3 (HER3)(adenocarcinoma), and erbB4 (HER4) (breast cancer).

Tissue- or lineage-specific cancer antigens also include cell-associatedproteins such as tyrosinase, melan-A/MART-1, tyrosinase related protein(TRP)-1/gp75 (malignant melanoma), polymorphic epithelial mucin (PEM)(breast tumors), and human epithelial mucin (MUC1) (breast, ovarian,colon and lung cancers).

Tissue- or lineage-specific cancer antigens also include secretedproteins such as monoclonal immunoglobulin (multiple myeloma andplasmacytoma), immunoglobulin light chains (Multiple Mycloma),α-fetoprotcin (liver carcinoma), kallikrcins 6 and 10 (ovarian cancer),gastrin-releasing peptide/bombesin (lung carcinoma), and prostatespecific antigen (prostate cancer).

Still other cancer antigens are cancer testis (CT) antigens that areexpressed in some normal tissues such as testis and in some casesplacenta. Their expression is common in tumors of diverse lineages andas a group the antigens form targets for immunotherapy. Examples oftumor expression of CT antigens include MAGE-A1, -A3, -A6, -A12, BAGE,GAGE, HAGE, LAGE-1, NY-ESO-1, RAGE, SSX-1, -2, -3, -4, -5, -6, -7, -8,-9, HOM-TES-14/SCP-1, HOM-TES-85 and PRAME. Still other examples of CTantigens and the cancers in which they are expressed include SSX-2, and-4 (Neuroblastoma). SSX-2 (HOM-MEL-40), MAGE. GAGE, BAGE and PRAME(Malignant melanoma), HOM-TES-14/SCP-1 (Meningioma), SSX-4(Oligodendrioglioma), HOM-TES-14/SCP-1, MAGE-3 and SSX-4 (Astrocytoma),SSX member (Head and neck cancer, ovarian cancer, lymphoid tumors,colorectal cancer and breast cancer), RAGE-1, -2, -4, GAGE-1, -2, -3,-4, -5, -6, -7 and -8 (Head and neck squamous cell carcinoma (HNSCC)),HOM-TES14/SCP-1, PRAME, SSX-1 and CT-7 (Non-Hodgkin's lymphoma), andPRAME (Acute lymphoblastic leukemia (ALL), acute myelogenous leukemia(AML) and chronic lymphocytic leukemia (CLL)).

Other cancer antigens are not specific to a particular tissue or celllineage. These include members of the carcinoembryonic antigen (CEA)family: CD66a, CD66b, CD66c, CD66d and CD66e. These antigens can beexpressed in many different malignant tumors and can be targeted byimmunotherapy. Still other cancer antigens are viral proteins and theseinclude Human papilloma virus protein (cervical cancer), and EBV-encodednuclear antigen (EBNA)-1 (lymphomas of the neck and oral cancer). Stillother cancer antigens are mutated or aberrantly expressed molecules suchas but not limited to CDK4 and beta-catenin (melanoma).

Autoimmune Diseases

Also contemplated are methods of modulating immune responses in asubject by administering to such subject the glycolipid describedherein. The subject may be one having or likely to develop an aberrantimmune response. The aberrant immune response may be an enhanced immuneresponse and the glycolipid acts to down-regulate the immune response.Enhanced immune responses are typically associated with inflammatoryconditions, such as but not limited to autoimmune diseases. Autoimmunediseases, autoimmune disorders and autoimmune conditions are usedinterchangeably herein.

Accordingly, the compositions comprising glycolipid as a single agent orin combination with an antigen other than lipidated PSA, may be used tomodulate (e.g., down-regulate) certain immune responses in subjectshaving or at risk of developing autoimmune diseases. As will beunderstood by those of ordinary skill in the art, subjects havingautoimmune diseases typically experience one or more “events” orrecurrences associated with the autoimmune disease. For example, asubject having inflammatory bowel disease may experience temporallyisolated attacks of the disease, characterized by the presence ofsymptoms or increased severity of symptoms. The invention contemplatesthat the compositions may be used in such subjects to reduce thelikelihood of such future recurrences of the disease or to reduce theseverity of symptoms associated with the disease (e.g., pain, fever,discomfort, fatigue, etc.). Thus, the compositions may be administeredprior to such recurrence, and in this manner may be chronicallyadministered, optionally at a regular frequency. Examples include once aday, once every 2, 3, 4, 5 or 6 days, or once a week, etc. The inventionalso contemplates that the compositions may be administered to thesubject during a recurrence in order to reduce the severity of symptomsor shorten the time of the recurrence.

Thus, as an example, the invention provides a method comprisingadministering to a subject at risk of a recurrence of a conditionassociated with inflammation an effective amount of the glycolipid as asingle agent or in combination with an antigen such as or alternative toPSA or lipidated PSA. The method may reduce the likelihood of arecurrence of the condition or may reduce the frequency of futurerecurrences. The method may reduce the severity of symptoms associatedwith the condition, whether such symptoms are present in the firstmanifestation, in a recurrence, or chronically.

Autoimmune diseases are known in the art. Examples of autoimmunediseases include but are not limited to multiple sclerosis, inflammatorybowel disease including Crohn's Disease and ulcerative colitis,rheumatoid arthritis, psoriasis, type I diabetes, uveitis, Celiacdisease, pernicious anemia, Srojen's syndrome. Hashimoto's thyroiditis,Graves' disease, systemic lupus erythamatosis, acute disseminatedencephalomyelitis, Addison's disease, Ankylosing spondylitis,Antiphospholipid antibody syndrome. Guillain-Barre syndrome, idiopathicthrombocytopenic purpura, Goodpasture's syndrome, Myasthenia gravis,Pemphigus, giant cell arteritis, aplastic anemia, autoimmune hepatitis,Kawaski's disease, mixed connective tissue disease, Ord throiditis,polyarthritis, primary biliary sclerosis, Reiter's syndrome, Takaysu'sarteritis, vitiligo, warm autoimmune hemolytic anemia, Wegener'sgranulomatosis, Chagas' disease, chronic obstructive pulmonary disease,and sarcoidosis.

In important embodiments, the autoimmune disease is multiple sclerosis.In other important embodiments, the autoimmune disease is aninflammatory bowel disease including but not limited to ulcerativecolitis and Crohn's disease. In other embodiments, the autoimmunedisease may be rheumatoid arthritis or type I diabetes.

In some instances, the compositions of the invention may be administeredto a subject who has yet to manifest an autoimmune disease (includingsymptoms thereof) yet is at risk of developing such as disease based ona known genetic or familial predisposition. Such a subject may have oneor more family members that are afflicted with the disease.

In some instances, the compositions of the invention are administered tosubject having or at risk of developing graft-versus-host disease.Administration may occur prior to, during and/or after transplantationof an organ or tissue (including blood or a blood product) into thesubject.

In still other instances, the compositions may be administered tosubjects having or at risk of developing a conditions associated withinflammation.

As an example, the composition may be administered to a subject havingasthma. As will be understood in the art, subjects having asthmatypically experience asthmatic attacks or events characterized byimpaired breathing. The invention contemplates that the compositionsdescribed herein may be administered acutely (e.g., a single large dose)or chronically (e.g., repeated, smaller doses) to asthmatic subjects.Accordingly, in some instances, the compositions may be administeredprior to an asthmatic attack in order to prevent the occurrence of theattack, reduce the frequency of attacks, and/or to lessen the severityof the attack. In some instances, the compositions may be administeredduring an attack in order to reduce its severity and/or reduce itsduration.

Another condition associated with inflammation is a post-surgicaladhesion. The invention contemplates administration of the compositionsdescribed herein to subjects having or at risk of developing apost-surgical adhesion. The compositions may be administered prior to,during, and/or immediately following surgery, or any combination thereofincluding but not limited to prior to and during surgery, in order toprevent the occurrence of such adhesions and/or reduce their severity.The compositions may be administered repeatedly following surgery,including for example every day, every two days, every three days, etc.for a week, two weeks, three weeks, a month, or several monthspost-surgery.

Another condition associated with inflammation is an abscess, includingbut not limited to an abdominal abscess as may occur upon leakage ofintestinal contents into the peritoneum. In these instances, thesubjects being treated may also be administered anti-bacterial agentssuch as antibiotics.

Thus, as another example, a method is provided that comprisesadministering to a subject having or at risk of developing an abscess aneffective amount of the glycolipid (separate from the polysaccharidecomponent of PSA) or compositions thereof. In some embodiments, theglycolipid is administered prior to development of an abscess and/orprior to the manifestation of symptoms associated with an abscess. Insome embodiments, the glycolipid is administered after an abscess hasbeen detected or diagnosed and/or after symptoms associated with anabscess are manifested.

A subject intends any subject that would benefit from administration ofa composition of the invention or that could be administered thecomposition of the invention. In important embodiments, the subject is ahuman subject. The subject may also be a companion animal such as a dogor cat, agricultural livestock such as horses, cattle, pigs, sheep,etc., laboratory animals such as mice, rats, rabbits, monkeys, etc., oranimals such as those maintained in zoos or otherwise in captivity.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular condition beingtreated, the severity of the condition being treated, and the dosagerequired for therapeutic efficacy. The methods of the invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical, nasal, inhalation (e.g., inhaler or nebulization), orparenteral routes. The term “parenteral” includes subcutaneous,intravenous, intramuscular, intraperitoneal, or infusion.

Formulations

When administered, the agents (e.g., glycolipids) of this disclosure areformulated as pharmaceutically acceptable compositions or preparations.Such compositions or preparations may routinely contain pharmaceuticallyacceptable carriers, concentrations of salt, buffering agents,preservatives, other immune modulators, and optionally other therapeuticagents. The term “pharmaceutically-acceptable carrier” as used herein,and described more fully below, means one or more compatible solid orliquid filler, diluents or encapsulating substances which are suitablefor administration to a human or other animal. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active agent(s) is combined to facilitate administration,long-term storage, stability and the like. The active agents of thepresent invention may be comingled with the other components of thepharmaceutical compositions, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The pharmaceutical compositions may be presented in unit dosage form andmay be prepared by any of the methods known in the art of pharmacy. Allmethods include the step of bringing the active agents into associationwith a carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the active agent(s) into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product. Compositions suitable for oral administration may bepresented as discrete units, such as capsules, tablets, lozenges, eachcontaining a predetermined amount of the active agent. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as a syrup, elixir or an emulsion. Solid forms may becoated for example enterically coated.

The active agent(s) may be administered per se (neat) or in the form ofa pharmaceutically acceptable salt. Pharmaceutically acceptable saltsmay be used for in vivo applications as well as in vitro applications.Non-pharmaceutically acceptable salts may be used to preparepharmaceutically acceptable salts thereof and are not excluded from thescope of the invention. Pharmaceutically acceptable salts include, butare not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicyclic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, pharmaceutically acceptable salts can beprepared as alkyline metal or alkyline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt 1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active agent(s), which canbe isotonic with the blood of the recipient. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Carrier formulations suitable forsubcutaneous, intramuscular, intraperitoneal intravenous, etc.administrations may be found in Remington's Pharmaceutical Sciences,Mack Publishing Company. Easton, Pa.

In some embodiments, the glycolipid is formulated with a detergent suchas but not limited to Tween or a bile salt such as but not limited todeoxycholate (e.g., sodium deoxycholate) in order to limit or preventaggregation. Such detergent or bile salt may be used at a lowconcentration such that it is still pharmaceutically acceptable. Forexample, it may be present at about or less than 0.0001%, 0.0005%,0.001%, 0.002%, 0.005%, 0.01%, 0.02%, 0.05%, 0.07%, 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or more.

The pharmaceutical preparations, as described above, are administered ineffective amounts. For therapeutic applications, it is generally thatamount sufficient to achieve a medically desirable result. In general, atherapeutically effective amount is that amount necessary to delay theonset of, inhibit the progression of, or halt altogether the particularcondition being treated, including reducing the likelihood, frequencyand/or severity of a recurrence of the condition. As an example, theeffective amount may be that amount which serves to reduce, alleviate,or delay the onset of the symptoms (e.g., pain, fever, etc.) of thedisorder being treated or prevented. The effective amount will dependupon the mode of administration, the particular condition being treatedand the desired outcome. It will also depend upon the stage of thecondition, the severity of the condition, the age and physical conditionof the subject being treated, the nature of concurrent therapy (if any),the duration of the treatment, the specific route of administration andlike factors within the knowledge and expertise of the medicalpractitioner. For prophylactic applications, it is that amountsufficient to delay the onset of, inhibit the progression of, or haltaltogether the particular condition being prevented, and may be measuredby the amount required to prevent the onset of symptoms.

Generally, doses of active agent(s) of the present invention may be fromabout 0.01 mg/kg per day to 1000 mg/kg per day, preferably from about0.1 mg/kg to 200 mg/kg and most preferably from about 0.2 mg/kg to about20 mg/kg, in one or more dose administrations daily, for one or moredays. It is expected that doses ranging from 1-500 mg/kg, and preferablydoses ranging from 1-100 mg/kg, and even more preferably doses rangingfrom 1-50 mg/kg, will be suitable. The preferred amount can bedetermined by one of ordinary skill in the art in accordance withstandard practice for determining optimum dosage levels of the agent. Itis generally preferred that a maximum dose is the highest safe doseaccording to sound medical judgment be used.

In some instances, the total daily dose for a human subject may rangefrom about 50-100 micrograms of the glycolipid.

The pharmaceutical preparation may be administered alone or inconjunction with one or more other active agents.

The pharmaceutical preparation may be used or administered inconjunction with active agents that are suitable for autoimmunedisorders such as multiple sclerosis, Crohn's disease, ulcerativecolitis, asthma, rheumatoid arthritis, and the like.

An example of such agents include anti-inflammatory agents. Examplesinclude steroids and corticosteroids such as cortisone; non-steroidalanti-inflammatory drugs such as aspirin, salsalate, celecoxib,diclofenac, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac,nabumetone, naproxen, oxaprozin, piroxicam, sulindac, and tolmetin;aminosalicylates such as sulfasalazine and 5-aminosalicylates includingmesalamine, balsalazide, and olsalazine; azathioprine; mercaptopurine;cyclosporine; beta interferons; glatiramer acetate; dimethyl fumarate;fingolimod; mitoxantrone; disease-modifying antirheumatic drugs (DMARDs)such as methotrexate, leflunomide, hydroxychloroquine and sulfasalazine.

Another example of such agents include antibodies or antibody fragments.Examples include TNF alpha inhibitors such as infliximab (Remicade),adalimumab (Humira), and golimumab (Simponi); natalizumab (Tysabri),vedolizumab (Entyvio); ustekinumab (Stelara); abatacept (Orencia);anakinra (Kineret); certolizumab (Cimzia), etanercept (Enbrel),rituximab (Rituxan), tocilizumab (Actemra), and tofacitinib (Xeljanz).

The invention contemplates that the combined use of glycolipid togetherwith standard treatments such as those recited above will allow a lowerdose of the standard treatment to be used for the same or bettertherapeutic effect, and/or will result in reduced incidence and/orseverity of side effects associated with such standard treatments.

In some embodiments, the subject is also administered an anti-bacterialagent such as an antibiotic. In one embodiment the pharmaceuticalpreparation is formulated or given in conjunction with one or moreanti-bacterial agents including antibiotics selected from the groupconsisting of penicillin G, penicillin V, ampicillin, amoxicillin,bacampicillin, cyclacillin, epicillin, hetacillin, pivampicillin,methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin,flucloxacillin, carbenicillin, ticarcillin, avlocillin, mezlocillin,piperacillin, amdinocillin, cephalexin, cephradine, cefadoxil, cefaclor,cefazolin, cefuroxime axetil, cefamandole, cefonicid, cefoxitin,cefotaxime, ceftizoxime, cefmnenoxine, ceftriaxone, moxalactam,cefotetan, cefoperazone, ceftazidime, imipenem, clavulanate, timentin,sulbactam, neomycin, erythromycin, metronidazole, chloramphenicol,clindamycin, lincomycin, vancomycin, trimethoprim-sulfamethoxazole,aminoglycosides, quinolones, tetracyclines and rifampin.

The following Examples are included for purposes of illustration and arenot intended to limit the scope of the invention.

EXAMPLES Example 1. Isolation of Glycolipid

Briefly, B. fragilis was grown in anaerobic conditions. The capsularcomplex from B. fragilis was isolated with hot phenol/water extraction.The polysaccharide fraction was precipitated with ethanol after DNAse.RNase and pronase treatments. The precipitate was subjected to sizeexclusion chromatography in order to separate polysaccharideconstituents. The fractions of interest were analyzed and pooled, thendialyzed and lyophilized. The purity of glycolipid can be assessed bynuclear magnetic resonance spectroscopy and mass spectroscopy.

The isolation and purification process is provided below in greaterdetail.

The B. fragilis delta44 mutant strain was derived experimentally fromstrain 9343 and upon further characterization it was found toover-express PSA relative to PSB. Delta44 was plated onto a blood agarplate and grown overnight at 37° C. A swab from a heavily colonizedplate was sub-cultured into a 500 ml starter culture of peptone yeastbroth. The starter culture was inoculated into 16 liter culture of thesame media and pH was titrated to neutrality with 5M NaOH. An anaerobicgas mix was bubbled into the sealed culture.

After an overnight culture maintained at pH 7, bacteria were checked byGram stain and subculture. Organisms were collected by centrifugation at8,000×g for 20 minutes. Bacterial pellets were washed two times withsaline yielding approximately one liter of bacterial pellet.

The bacterial pellet was suspended in 68° C. melted crystalline phenolto a final concentration of phenol of about 37% v/v (yielding aphenol/water preparation) and mixed for 30 minutes at 68° C. followed bystirring at 4° C. for 48 hours. The phenol/water preparation wasaliquoted into glass bottles which were then centrifuged at 1500 rpm.The upper water layer was harvested. Any residual phenol contained inthe harvested aqueous phase was extracted with an equal volume of ethylether. The ether phase was then removed using a separatory funnel andany residual ether in the aqueous phase was evaporated, yielding thefinal aqueous phase from the phenol/water preparation.

The aqueous phase was dialyzed versus water with multiple changes over 5days at 4° C. and subsequently lyophilized until it was nearly dry(approximately 5 ml water remaining). A solution of 0.05M Tris withmagnesium, calcium and sodium azide (total volume 61 ml) was added tothe lyophilized product to bring the total volume to about 66 ml.

To the dissolved product was added 10 ml of Tris buffer with DNase (0.07mg/ml) and RNase (0.33 mg/ml). The entire suspension was filteredthrough a 0.45 micron filter and the filtrate was stirred at 37° C. TheDNase/RNase treatment was repeated by adding fresh enzymes to themixture, at similar concentrations, and stirred for two hours.

The mixture was then combined with 25 mg pronase in 10 mlTris/magnesium/calcium solution, and the mixture stirred for 24 hours at37° C. This step was repeated.

The polysaccharide fraction was precipitated by adding 5 volumes ofethanol at 4° C. to the mixture. The solution was then centrifuged at12,000×g for 30 minutes to pellet the polysaccharide fraction. Thesupernatant was removed and the pellet was resuspended in 392 ml type 1H₂O.

The dissolved fraction was then dialyzed against two changes of 16liters type 1 H₂O at 4° C. The volume was reduced by lyophilization toapproximately 50 mls.

Twenty ml aliquots were chromatographed on a 5×200 cm column of S400suspended in PBS and 1% sodium deoxycholate, and fractions werecollected. Fractions were tested by double diffusion in agar with anantibody that reacts with both lipidated and non-lipidated PSA todetermine where lipidated PSA eluted. Aliquots were tested for UVabsorption at 280 nm and it was determined that these fractions had noUV absorbable material (e.g., no nucleic acids, no proteins).

Fractions were then pooled, concentrated and dialyzed against type 1 H₂Oon a Minitan concentrator (Millipore) with 10,000 mw cutoff membranesuntil conductivity of 100 ml was less than 50 ρS. The final product wasthen lyophilized.

Polysaccharide and glycolipid purity and structure was determined byproton nuclear magnetic resonance spectroscopy on a 600 MHz spectrometerand mass spectroscopy. For MALDI-TOF-TOF and LC-MS analysis, the samplewas resuspended to 10 μg/p L in 2% acetic acid and heated at 90 degreesfor 90 minutes. For MALDI-TOF-TOF analysis, samples were mixed 1:1 to 1%matrix (CHCA or DHB) solution and directly loaded to stainless steelMALDI plate. For LC-MS analysis, samples did or did not undergoliquid-liquid extraction (chloroform-water) step, and then were driedand resuspended in 50:50 isopropanol:acetonitrile and injected. The acidtreatment in this step results in the cleavage of the oligosaccharidecore unit from the acyl-substituted diglucosamine.

An animal model of multiple sclerosis (EAE) may be used to study theimmunological activity of glycolipid in vivo. In this model, mice aretreated with glycolipid (on the order of about 75-100 μg per mouse) orcontrol (saline, PBS) every three days starting 6 days before EAEinduction. Mice are challenged subcutaneously with 250 μg of MOG₃₃₋₅₅(Peptides International) in 200 μl of complete Freund's adjuvant(Sigma). On days 0 and 2 after challenge, mice receive intraperitonealinjections of 250 ng of Bordetella pertussis toxin (List BiologicalLaboratories). Disease is scored on an established 0 to 5 scale, with 5being advanced neurological disease. Mice are monitored and scored dailyfor disease progression.

Example 2. Preparation of Lipids for Chemical Analysis

For the experiments shown in FIGS. 9-13 , the following procedure wasused to prepare glycolipids. Semi-purified B. fragilis glycolipids orovernight-grown bacterial pellet was resuspended in 100 mM aceticacid/sodium acetate buffer (pH 4.5) with 1% sodium dodecylsulfate (SDS)solution at 1-10 mg/mL concentration. The mixture was heated at 95° C.for 30 minutes and cooled. Solution was added with the same volume ofchloroform and methanol, shaken well and spun at 1000 g for 10 mins forphase separation. Lower organic phases, containing hydrolyzed GLA, werecollected and dried under nitrogen stream and resuspended in 65%isopropanol solution. These samples were injected to reverse-phase HPLC(Phenomenex Kintex C8, 150 mm×4.6 mm×5 um) with the 65-90% gradient ofIPA with 10 mM ammonium formate. Fractions were collected, re-analyzedwith LC-MS for quantitation and resuspended in DMSO or 0.05% tween-20PBS solution for in vitro/in vivo experiments.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routing experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an.” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one. A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising.” “including,” “carrying.” “having,”“containing,” “involving.” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofpatent Examining Procedures. Section 2111.03.

1. A glycolipid comprising a tri-acylated, tetra-acylated orpenta-acylated diglucosamine conjugated to an oligosaccharide. 2.(canceled)
 3. The glycolipid of claim 1, wherein the tri-acylated,tetra-acylated or penta-acylated diglucosamine comprises acyl chainsranging in length from 14-17 carbons.
 4. (canceled)
 5. The glycolipid ofclaim 1, substantially free of other components found in a B. fragiliscapsule, substantially free of LPS, substantially free ofpolysaccharide, in purified form, in an isolated form, and/or in alyophilized form. 6-9. (canceled)
 10. The glycolipid of claim 1, in amicelle form or a liposome form. 11-12. (canceled)
 13. The glycolipid ofclaim 1, obtained or derived from B. fragilis cells that overexpress PSArelative to polysaccharide B (PSB), PSC, PSD, PSE, PSF, PSG and PSH. 14.The glycolipid of claim 1, wherein at least one acyl chain isunmodified. 15-17. (canceled)
 18. The glycolipid of claim 1, wherein atleast one acyl chain is C16:0-OH; C17:0-OH; C14:0; C15:0; N-substituted;or O-substituted. 19-24. (canceled)
 25. The glycolipid of claim 1,wherein the diglucosamine is phosphorylated.
 26. The glycolipid of claim1, wherein the diglucosamine is conjugated to the oligosaccharide by anacid-labile bond.
 27. (canceled)
 28. The glycolipid of claim 1, whereinthe oligosaccharide contains 3-10 sugars.
 29. The glycolipid of claim 1,wherein the oligosaccharide comprises galactose and/or glucose and/orfucose residues and/or 1 to 2 KDO residues.
 30. (canceled)
 31. Acomposition comprising the glycolipid of claim 1, and any one selectedfrom the group consisting of: (i) a detergent or a bile salt; (ii) anon-naturally occurring preservative; (iii) a non-naturally occurringstabilizer; (iv) human albumin, phenol, glycerin or glycine; (v)thimerosal, aluminum hydroxide, benzethonium chloride, formaldehyde,formalin, glutaraldehyde, potassium phosphate, aluminum potassiumsulfate, bovine extract, calf serum, ammonium sulfate, aluminumphosphate, non-human cells, Vero (monkey kidney) cells, human cells,MRC-5 (human diploid) cells, and/or MRC-5 cellular proteins; (vi) alipidated polysaccharide A (PSA), wherein the lipidated polysaccharide A(PSA) and the glycolipid are in a 4:1 to 20:1 ratio (w/w); (vii) alipidated polysaccharide A (PSA), wherein there is at least 0.5% (w/w)of the glycolipid; (viii) an antigen; and (ix) an antigen and alipidated PSA.
 32. The composition of claim 31, wherein the detergent orbile salt is present at or less than 1%, 0.5% or 0.1% (weight ofdetergent or bile salt as a percentage of weight of glycolipid). 33-36.(canceled)
 37. The composition of claim 31, formulated for parenteraladministration or oral administration. 38-48. (canceled)
 49. Thecomposition of claim 31 wherein the antigen is a bacterial antigen,attenuated bacteria, a viral antigen, an attenuated virus, a fungalantigen, a mycobacterial antigen, a parasitic antigen, a cancer antigen,a human protein, or a human polysaccharide. 50-82. (canceled)
 83. Amicelle consisting essentially of glycolipid of claim
 1. 84. Acomposition comprising a micelle consisting essentially of glycolipid ofclaim 1 and a detergent or bile salt.
 85. The composition of claim 84,wherein the detergent or bile salt is present in a pharmaceuticallyacceptable amount.
 86. The composition of claim 84, wherein thecomposition is a pharmaceutical composition.
 87. The composition ofclaim 84, formulated for parenteral administration. 88-95. (canceled)